Fibrin-specificity of a plasminogen activator affects the efficiency of fibrinolysis and responsiveness to ultrasound: comparison of nine plasminogen activators in vitro.

In a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed. Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot. In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

Acceleration of fibrinolysis by high-frequency ultrasound: the contribution of acoustic streaming and temperature rise.

High-frequency ultrasound has been shown to accelerate enzymatic fibrinolysis. One of the supposed mechanisms of this effect is the enhancement of mass transport by acoustic streaming, i.e., ultrasound-induced macroscopic flow around the clot. In this study, which is aimed at further elucidating the mechanisms of the acceleration of fibrinolysis by ultrasound, we investigated whether ultrasound would accelerate fibrinolysis if the flow around the thrombus is already present, as may occur in vivo. The effect of the ultrasound-induced temperature rise was also studied. In a model of a plasma clot submerged in plasma, containing tissue-type plasminogen activator, mild stirring of the outer plasma producing a shear rate of 40 seconds(-1) at the surface of the clot resulted in a two-fold acceleration of lysis. A similar effect was obtained with ultrasound (1 MHz, 2 W/cm(2)). Furthermore, if ultrasound was applied together with stirring, only 30% acceleration by ultrasound was documented, fully attributable to the concomitant temperature rise. In a model with tissue-type plasminogen activator incorporated throughout a plasma clot, the effect of ultrasound (two-fold shortening of lysis time) was fully attributable to the concomitant temperature rise of a few degrees. We concluded that the acceleration of enzymatic plasma clot lysis by high-frequency ultrasound in the models we used can be largely explained by a combination of the effects of heating and acoustic streaming, equivalent to mild stirring. The thermal effects can hardly be utilized in vivo due to the danger of tissue overheat. The therapeutic advantage of transcutaneous high-frequency ultrasound as an adjunct to thrombolytic therapy may appear limited to the situations where there is no flow in the direct environment of the thrombus.

Development of standard measurement methods for essential properties of ultrasound therapy equipment.

In a European collaborative project, partly funded by the EC Community Bureau of Reference (BCR), reliable methods of measurement for characterising the output and performance of ultrasound physiotherapy equipment have been developed. Experimental investigations using miniature hydrophones to scan the distribution of pressure in therapeutic fields have been undertaken in combination with theoretical simulations of the sound fields. Important parameters such as Beam Cross Sectional Area (BCSA), Effective Radiating Area (ERA) and Beam Nonuniformity Ratio (BNR) (characterising "Hot-spots": potentially harmful to patients) have been redefined, and these new definitions have been incorporated in a revision of IEC 150:1963. The reproducibility and accuracy of measurements of ERA based on these procedures are presented in detail for a variety of therapy fields. Furthermore, it is shown that the value of the BNR for any treatment head should not exceed 8. Values of effective intensity derived using the new procedures are shown to be significantly higher than those obtained using FDA (USA) definitions, a conclusion in agreement with the theoretical expectations. Measurements on four treatment heads were used to validate the procedures of the proposed revised standard. Values of ERA derived by the two laboratories were in agreement to within 2.5%.

Effect of therapeutic ultrasound on endochondral ossification.

The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (Isatp) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (Isata). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation.

Validated ultrasonic power measurements up to 20 W.

A project has been completed to develop reference methods for the measurement of ultrasonic power with a validated measurement uncertainty of < 7% at power levels of 1 to 20 W over the frequency range 1 to 3 MHz of collimated beams. The project is the result of collaborative research between the Physikalisch-Technische Bundesanstalt, Germany (PTB, DE), the National Physical Laboratory, UK (NPL, UK) and the Netherlands Organisation for Applied Scientific Research, Prevention and Health (TNO-PG, NL). The work has been undertaken under the 4th Framework Programme of the European Community (EC). Primary standard designs of radiation force balances based on both absorbing and reflecting targets have been constructed. To avoid heating effects, the measurements should be done relatively quickly (10 to 20 s). The methods have been validated using ultrasound (US) transducers that demonstrated an adequate short and long-term stability; a method to detect cavitation based on monitoring the acoustic signals produced by bubble oscillation and collapse has been confirmed. It has been shown that only the detection of the subharmonic can be used in practice as cavitation detector. Different procedures for obtaining degassed water have been investigated. A method showing significant promise to be used in a clinical or manufacturer's environment involves the addition of sodium sulphite (Na2SO3). During the validation process, commercially available radiation force balances and ultrasonic physiotherapy devices have also been evaluated. Limitations of current measurement methods and practices, including power measurements made on transducers exhibiting a diverging beam, have been identified. It has been shown that a reflecting target is not appropriate to measure powers of transducers with a ka-value < 30. Based on beam shape and target distance, it has been shown also that proper power measurements using a 45 degrees convex-conical reflecting target can never be performed for transducers with a ka-value < 17.4.

Characterising ultrasonic physiotherapy systems by performance and safety now internationally agreed.

The IEC 1689 standard specifies: methods of measurement, characterisation of the output performance, requirements for safety due to the ultrasonic field generated, characteristics to be declared by manufacturers and acceptance criteria for aspects of performance. For the characterisation, reference as well as routine testing methods are described. Important parameters such as effective radiating area (AER), beam nonuniformity ratio (RBN) (characterising 'hot-spots': potentially harmful to patients) and beam type have been re-defined. The quality, reproducibility and accuracy of measurements of AER are significantly improved compared to those in IEC 150 (which is now replaced) and the FDA 1050.10 (USA) standards. As a result, values of effective intensity are shown to be significantly higher than those obtained using the FDA 1050.10 (USA) definitions. As it may be expected that the new standard will be implemented worldwide, this problem is just a matter of time. The standard discussed above specifies the need for acoustic power measurement with an accuracy of more than 15%. However, several investigations carried out over the last ten years show a lack of proper facilities to calibrate the acoustic output power at physiotherapy levels. A project to investigate the complications and to produce definitive guidance for therapy-level power measurements is funded by the EC and has been started recently. Future activities will be undertaken to estimate the expected temperature rise in tissue due to the ultrasonic field. Such a figure may result in an improved judgement of the ultrasonic safety aspects of physiotherapy systems.